Central Linker Region Research Articles

Identification and Characterization of EhCaBP2: A SECOND MEMBER OF THE CALCIUM-BINDING PROTEIN FAMILY OF THE PROTOZOAN PARASITE ENTAMOEBA HISTOLYTICA

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.

Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II.

Backbone dynamic properties of the central linker region of calcium-calmodulin in 35% trifluoroethanol

The backbone dynamic properties of uniformly 15N-labeled calcium-saturated calmodulin (Ca 2+-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by 15N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca 2+-CaM, which is a long, solvent-exposed α-helix in the crystal structure but is known to be partially unwound and flexible in solution. 15N T 1, T 2, and 15N–{ 1H} NOE values were determined for both Ca 2+-CaM in H 2O and Ca 2+-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78–Glu80 of 0.29, 0.17, and 0.27 in H 2O and 0.82, 0.66, and 0.64 in 35% TFE. However, 15N–{ 1H} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78–Glu80 15N–{ 1H} NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca 2+-CaM in 35% TFE, with motional correlation times of ∼10 and ∼9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca 2+-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.

A Novel Nuclear Export Signal in Smad1 Is Essential for Its Signaling Activity

To investigate the subcellular distributions of Smad proteins, the intracellular mediators of transforming growth factor-beta family cytokines, we examined their sequences for nuclear export signals (NES). We found a leucine-rich NES-like motif (termed NES2) in the central linker region of the receptor-regulated Smads that is absent from the other two classes of Smads (Co-Smads and I-Smads). In microinjection assays, NES2 peptide caused nuclear export of a fused glutathione S-transferase protein. Mutations in NES2 converted Smad1 from an even distribution throughout the cells into an exclusive nuclear localization in both transiently and stably expressing cell lines, and this nuclear enrichment was more pronounced than that induced by mutations in NES1. Furthermore, overexpression of CRM1, the cellular export receptor, transforms Smad1 into a mostly cytoplasmic profile by enhancing its nuclear export. The Smad1 NES2 mutant but not the Smad1 NES1 mutant is mostly resistant to this cytoplasmic targeting, indicating that NES2, not NES1, is the major target for CRM1 in Smad1. We further confirmed the functionality of NES2 by a heterokaryon assay. The Smad1 NES1 mutant displays good ligand responsiveness and moderately lowered transcriptional activity compared with wild type Smad1. In contrast, the Smad1 NES2 mutant shows a severe disruption in reporter gene activation, minimal response to bone morphogenetic protein stimulation, and significantly lowered bone morphogenetic protein-induced phosphorylation, which may be the reason for its deficient transcription activity. Thus, we have defined a major NES in Smad1 that is essential for its ligand-induced coupling with cell surface receptors and hence, transcriptional activity. Our study, along with recent studies of the nucleocytoplasmic shuttling of Smad2 and Smad3 proteins, demonstrate that continued nucleocytoplasmic shuttling is a common requisite for the active signaling of R-Smads. Although conserved in other R-Smads such as Smad3, NES2 is not functional in these R-Smads because CRM1 overexpression fails to target them to cytoplasm. Possible reasons for this discrepancy are discussed.

Isoform Heterogeneity of the Human Gephyrin Gene (GPHN), Binding Domains to the Glycine Receptor, and Mutation Analysis in Hyperekplexia

Gephyrin (GPHN) is an organizational protein that clusters and localizes the inhibitory glycine (GlyR) and GABAA receptors to the microtubular matrix of the neuronal postsynaptic membrane. Mice deficient in gephyrin develop a hereditary molybdenum cofactor deficiency and a neurological phenotype that mimics startle disease (hyperekplexia). This neuromotor disorder is associated with mutations in the GlyR alpha1 and beta subunit genes (GLRA1 and GLRB). Further genetic heterogeneity is suspected, and we hypothesized that patients lacking mutations in GLRA1 and GLRB might have mutations in the gephyrin gene (GPHN). In addition, we adopted a yeast two-hybrid screen, using the GlyR beta subunit intracellular loop as bait, in an attempt to identify further GlyR-interacting proteins implicated in hyperekplexia. Gephyrin cDNAs were isolated, and subsequent RT-PCR analysis from human tissues demonstrated the presence of five alternatively spliced GPHN exons concentrated in the central linker region of the gene. This region generated 11 distinct GPHN transcript isoforms, with 10 being specific to neuronal tissue. Mutation analysis of GPHN exons in hyperekplexia patients revealed a missense mutation (A28T) in one patient causing an amino acid substitution (N10Y). Functional testing demonstrated that GPHNN10Y does not disrupt GlyR-gephyrin interactions or collybistininduced cell-surface clustering. We provide evidence that GlyR-gephyrin binding is dependent on the presence of an intact C-terminal MoeA homology domain. Therefore, the N10Y mutation and alternative splicing of GPHN transcripts do not affect interactions with GlyRs but may affect other interactions with the cytoskeleton or gephyrin accessory proteins.

Topological analysis and role of the transmembrane domain in polar targeting of PilS, a Pseudomonas aeruginosa sensor kinase.

In Pseudomonas aeruginosa, synthesis of pilin, the major protein subunit of the pili, is regulated by a two-component signal transduction system in which PilS is the sensor kinase. PilS is an inner membrane protein found at the poles of the bacterial cell. It is composed of three domains: an N-terminal hydrophobic domain; a central cytoplasmic linker region; and the C-terminal transmitter region conserved among other sensor kinases. The signal that activates PilS and, consequently, pilin transcription remains unknown. The membrane topology of the hydrophobic domain was determined using the lacZ and phoA gene fusion approach. In this report, we describe a topological model for PilS in which the hydrophobic domain forms six transmembrane helices, whereas the N- and C-termini are cytoplasmic. This topology is very stable, and the cytoplasmic C-terminus cannot cross the inner membrane. We also show that two of the six transmembrane segments are sufficient for membrane anchoring and polar localization of PilS.

Involvement of the Carboxy-Terminus Region of the Dihydropyridine Receptor β1a Subunit in Excitation-Contraction Coupling of Skeletal Muscle

Skeletal muscle knockout cells lacking the β subunit of the dihydropyridine receptor (DHPR) are devoid of slow L-type Ca 2+ current, charge movements, and excitation-contraction coupling, despite having a normal Ca 2+ storage capacity and Ca 2+ spark activity. In this study we identified a specific region of the missing β1a subunit critical for the recovery of excitation-contraction. Experiments were performed in β1-null myotubes expressing deletion mutants of the skeletal muscle-specific β1a, the cardiac/brain-specific β2a, or β2a/ β1a chimeras. Immunostaining was used to determine that all β constructs were expressed in these cells. We examined the Ca 2+ conductance, charge movements, and Ca 2+ transients measured by confocal fluo-3 fluorescence of transfected myotubes under whole-cell voltage-clamp. All constructs recovered an L-type Ca 2+ current with a density, voltage-dependence, and kinetics of activation similar to that recovered by full-length β1a. In addition, all constructs except β2a mutants recovered charge movements with a density similar to full-length β1a. Thus, all β constructs became integrated into a skeletal-type DHPR and, except for β2a mutants, all restored functional DHPRs to the cell surface at a high density. The maximum amplitude of the Ca 2+ transient was not affected by separate deletions of the N-terminus of β1a or the central linker region of β1a connecting two highly conserved domains. Also, replacement of the N-terminus half of β1a with that of β2a had no effect. However, deletion of 35 residues of β1a at the C-terminus produced a fivefold reduction in the maximum amplitude of the Ca 2+ transients. A similar observation was made by deletion of the C-terminus of a chimera in which the C-terminus half was from β1a. The identified domain at the C-terminus of β1a may be responsible for colocalization of DHPRs and ryanodine receptors (RyRs), or may be required for the signal that opens the RyRs during excitation-contraction coupling. This new role of DHPR β in excitation-contraction coupling represents a cell-specific function that could not be predicted on the basis of functional expression studies in heterologous cells.

Simultaneous binding of drugs with different chemical structures to Ca2+-calmodulin: crystallographic and spectroscopic studies.

The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 A resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3"-(beta-chloroethyl)-2",4"-dioxo-3, 5"-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955-962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

Calmodulin: a versatile calcium mediator protein

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is < or = 10(-7) M and during activation it rises to approximately 10(-6) M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix-loop-helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an alpha-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the alpha-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.

Small-angle x-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly .alpha.-helical conformation

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin. A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.

Solution structure of calmodulin

Solution structures of calmodulin, a ubiquitous calcium-binding protein in all eukaryotic cells, and calmodulin-peptide complex have been studied by solution scattering techniques. Calmodulin in solution possesses an elongated dumbbell shape, generally consistent with the crystal structure. A target peptide, such as melittin, mastoparan or MLCK-I, induces a large, calciumdependent conformational change in calmodulin. The overall shape of calmodulin-peptide comlex is globular. Using of the deletion mutants of calmodulin, we have revealed that the central linker region retains a predominantly α-helical conformation even in solution. A twisting motion would be a key step for the flexibility, as well as the bending. The globular structure is not a only possible conformation for the calmodulin-peptide complex. Some peptides form dumbbellshaped complexes with calmodulin.